Schistosoma haematobium in susceptibility and molecular diversity of the snail hosts globosus and B. nyassanus Stauffer Jr, J.R.; Madsen, Henry; Webster, B.; Black, K.; Rollinson, D.; Konings, A.

Published in: Journal of Helminthology

DOI: 10.1017/S0022149X08056290

Publication date: 2008

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Citation for published version (APA): Stauffer Jr, J. R., Madsen, H., Webster, B., Black, K., Rollinson, D., & Konings, A. (2008). in Lake Malawi: susceptibility and molecular diversity of the snail hosts and B. nyassanus. Journal of Helminthology, 82(4), 377-382. https://doi.org/10.1017/S0022149X08056290

Download date: 25. sep.. 2021 Journal of Helminthology (2008) 82, 377–382 doi:10.1017/S0022149X08056290

Schistosoma haematobium in Lake Malawˆ i: susceptibility and molecular diversity of the snail hosts Bulinus globosus and B. nyassanus

J.R. Stauffer Jr1, H. Madsen2, B. Webster3, K. Black1, D. Rollinson3 and A. Konings4 1School of Forest Resources, Penn State University, University Park, PA 16802, USA: 2DBL Centre for Health Research and Development, Faculty of Life Science, University of Copenhagen, Thorvaldsenvej 57, 1871 Frederiksberg C, Denmark: 3The Natural History Museum, Cromwell Road, London SW7 5BD, UK: 4PO Box 13608, El Paso, TX 79913, USA

Abstract

Intermediate hosts of Schistosoma haematobium, the causative agent of urinary schistosomiasis, in Lake Malawˆ i include: Bulinus globosus, a member of the B. africanus group and B. nyassanus, a diploid member of the B. truncatus/tropicus complex. We compared genetic variability between isolates of S. haematobium from the southern part of the lake (Cape Maclear), where both B. globosus and B. nyassanus play a role as intermediate hosts, and isolates from the northern part, where only B. globosus is host. Data show that the S. haematobium isolates from these two areas of Lake Malawˆ i cannot be distinguished using nuclear or mitochondrial sequences and are capable of cross-infections.

Introduction Lake Malawˆ i. Extensive sampling efforts (Stauffer et al., 2006) yielded no infected B. nyassanus outside of Historically, Bulinus globosus was the only known snail Nankumba Peninsula; thus, we believed that open- host for Schistosoma haematobium in Lake Malawˆ i. This water transmission was limited to this area. The school- snail typically inhabits inland, slow-moving waters and aged children residing in Chembe Village (Nankumba limited areas within the lake that are protected and, Peninsula) had a very high prevalence of urinary therefore, it was suspected that S. haematobium trans- schistosomiasis (.87% in 1998; Madsen et al., 2001) mission in Lake Malawˆ i was limited to such habitats and currently the highest found in villages along (Stauffer et al., 1997). Beginning in the mid-1980s, reports Lake Malawˆ i (Stauffer et al., 2006). Therefore, we of schistosomiasis among tourists suggested that, for the postulated that the current high prevalence of human first time, transmission was occurring in the open waters infection at Chembe Village is linked to B. nyassanus of Lake Malawˆ i (Harries et al., 1986; Whitworth, 1993; being an intermediate host in this portion of Lake Pollner et al., 1994; Cetron et al., 1996). It was clear that Malawˆ i. the epidemiology of S. haematobium in Lake Malawˆ i was changing, and the open waters of Lake Malawˆ i were no The host specificity of S. haematobium can be divided as longer free from urinary schistosomes. follows: strains most closely adapted to B. truncatus,a Madsen et al. (2001) discovered an endemic snail, tetraploid member of the B. truncatus/tropicus species Bulinus nyassanus, infected with human schistosomes in complex (North Africa and West Africa), strains most the open waters of Nankumba Peninsula in southern closely adapted to members of the B. africanus species group (West Africa, East Africa and southern Africa), and strains most closely adapted to the B. forskalii *E-mail: [email protected] species group (Brown, 1994; Southgate et al., 2000; 378 J.R. Stauffer et al.

Rollinson et al., 2001). The finding of B. nyassanus, in a total reaction volume of 25 ml using Ready-to-go PCR endemic to Lake Malawˆ i and a diploid member of the B. Beads (Amersham Pharmacia Biotech, Buckinghamshire, tropicus/truncatus species group, as intermediate host in UK), each containing 1.5 units DNA Taq polymerase, Lake Malawˆ i was therefore unexpected. 10 mM Tris–HCl (pH 9), 50 mM KCl, 1.5 mM MgCl2, We compare the genetic variation between S. haemato- 200 mM of each dNTP and stabilizers, including bovine bium from the southern part of the lake (Cape Maclear), serum albumin (BSA), 0.4 mM of each primer and 2 mlof where both B. globosus and B. nyassanus are intermediate DNA (,10 ng). Thermal cycling in a Perkin Elmer 9600 hosts, and from the northern part (Likoma Island), and Thermal Cycler used the following PCR conditions: 5 min investigate the compatibility of the S. haematobium from denaturing at 958C: 40 cycles of 30 s at 958C, 30 s at 40– Likoma Island with B. nyassanus from Chembe Village. 558C, 1–2 min at 728C; followed by a final extension Because infected B. nyassanus were collected only around period of 7 min at 728C. PCR products were visualized on Nankumba Peninsula in Lake Malawˆ i, we concluded that a 0.8% ethidium bromide agarose gel and then purified B. globosus was the only intermediate host at Likoma using Qiagen PCR Purification Kits (Qiagen) according to Island (Stauffer et al., 2006). the manufacturer’s protocol. Sequencing was performed using Fluorescent Dye Terminator Sequencing Kits (Applied Biosystems, Foster City, USA) and the sequen- Methods cing reactions run on either an Applied Biosystems 377 or a 373A automated sequencer. PCR products were Bulinus globosus and B. nyassanus were collected from sequenced directly using the original PCR primers; and Cape Maclear (Nankumba Peninsula). Snails were placed for COX1, 28S and 18S, internal sequencing primers were in 6 ml of water in 12.5 ml beakers and exposed to light also used to obtain the full sequence of each fragment to stimulate cercarial shedding. Cercariae shed from each from both strands (table 2). The sequences were snail were collected and preserved in 99% ethanol for assembled and manually edited using Sequencher ver- DNA extraction. sion 4.5 (GeneCodes Corp., Ann Abor, USA) and submitted to EMBL/GenBank. Each DNA region from each sample was aligned using Sequencher and sequence DNA extraction, DNA sequencing and phylogenetic analysis differences were checked by visualization of the sequence Cercariae collected from three snails of each species were chromatograms. used for molecular analysis. The ethanol-preserved The 28S and COX1 sequences were aligned by eye cercariae were centrifuged in a 1.5-ml Eppendorf tube in Maclade 4.05 (Sinauer Associates, Sunderland, Con- at 1300 rpm for 3 min to collect the cercariae at the bottom of necticut, USA) to sequence alignments of several other the tube. Most of the ethanol was pipetted from the sample, Schistosoma species and strains. Phylogenetic analyses which was then left to air dry for 10–30 min, allowing any were conducted separately using a neighbour-joining residual ethanol to evaporate from the sample. Once (NJ) algorithm to examine the evolutionary distance dry, total genomic DNA was extracted from each sample relationships among the samples. using the DNeasyTM Tissue Kit (Qiagen, West Sussex, UK) according to the manufacturer’s protocol. To identify any genetic variation between the samples, Compatibility testing two nuclear DNA and four mitochondrial DNA partial regions were amplified by polymerase chain reaction Schistosoma haematobium eggs were obtained from urine (PCR), as shown in table 1. Amplifications were performed samples from 10–15 infected children from Cape Maclear

Table 1. DNA regions utilized in this study and the primers used to amplify them. COX1, 16S, 28S and 18S regions were amplified as they have proved useful in phylogenetic studies, and the variable regions ND1 and a small section between Proline and COX1 were amplified as a result of preliminary data from studies by Littlewood, Webster & Huyse (pers. comm.) on Schistosoma haematobium mitochondrial genomes.

Region Size (bp) Forward primer Reverse primer

Mitochondrial DNA COX1 1164 Cox1_Schist_50 Cox1_Schist_30 50-TCTTTRGATCATAAGCG-30 50-TAATGCATMGGAAAAAAACA-30 16S 897 16S_Schist_30 16S_Schist_50 50-GATAAGAACCAACCTGGC-30 50-CTCGATGTTGGCTTGTTG-30 ND1 464 S. haem_ND1_F S. haem_ND1R CACCTTTAGCTTTTATTGCATGG TTAGAATGCTTCCGGCGTTA Proline– 242 S. haem_Pro_COX1F S. haem_Pro_COX1R COX1 50-GAGTTTATAGTGAGTTGGTTAG-30 50-GAAAGCAAGCAGTCTTCTAAC-30 Nuclear DNA 28S 1110 LSU5 1500R 50-TAGGTCGACCCGCTGAAYTTAAGCA-30 50-CGAAGTTTCCCTCAGGATAGCAAC-30 18S 2552 WA WB 50-GCGAATGGCTCATTAAATCAG-30 50-GGAAGTAAAAGTCGTAACAAG-30 Snail host susceptibility 379

Table 2. Internal sequencing primers. and Likoma Island. Urine samples were washed three times in saline water and once in fresh water (filtered lake Primer Direction Sequence 50 –30 water; 20 mm filter). After sedimentation in conical flasks for 25 min in the dark, the supernatant in the absence of 28S sequencing light was discarded and the flask filled with more washing ECD2 R CTTGGTCCGTGTTTCAAGACGGG 300F F CAAGTACCGTGAGGGAAAGTTG medium (saline or filtered lake water). After washing in 1200R R CCGAAAGATGGTGAACTATGC filtered lake water, the eggs were transferred to Petri 1200F F CCCGAAAGATGGTGAACTATGC dishes and more filtered lake water added. Hatching was 18S sequencing stimulated by exposing the Petri dishes to direct sunlight. 600R R TCAGGCTCCCTCTCCGGA Miracidia were captured with a tapering glass pipette and 600F F AGGGTTCGATTCCGGAG each snail was exposed to 5–20 miracidia. The uninfected 1200F F CAGGTCTGTGATGCCC B. nyassanus and B. globosus that were exposed came from 1200R R GGGCATCACAGACCTG Chembe Village. We exposed 25 B. globosus and 25 B. COX1 sequencing nyassanus to miracidia from eggs collected from children CO1560 F TTTGATCGGAATTTTGGTAC CO1800 R CCAACCATAAACATGTGATG at Chembe Village and 25 B. globosus to miracidia from eggs from children at Likoma Island. Subsequently, we

Fig. 1. Neighbour joining (distance tree); 28S (nuclear DNA). 380 J.R. Stauffer et al. repeated the exposure of 25 B. nyassanus from Chembe was surprising, especially due to the fact that B. nyassanus Village to miracidia from eggs captured from Likoma (B. truncatus/tropicus complex) is an unusual intermedi- Island children. The exposed snails were kept in separate ate host for this parasite. Within the B. truncatus/tropicus beakers under light for 6 h to facilitate infection, and complex, most diploid species seem to be resistant to maintained in aquaria (Madsen et al., 2001). From 5 to 8 infection in nature (Brown, 1994), although B. liratus on weeks after exposure, snails were checked individually the island of Madagascar appears to be an exception once a week for cercarial release: snails were transferred to beakers as described above and exposed to indirect (Stothard et al., 2001). The fact that B. nyassanus is also sunlight for 2 h. The water in each beaker was visually susceptible is therefore another interesting exception to checked for the presence of cercariae. the rule, because it is diploid (Madsen et al., 2001), and is a species within the B. truncatus/tropicus complex (Jørgensen et al., 2007). Both B. tropicus and B. truncatus Results are known to occur in other parts of Malawˆ i and, given ˆ The 28S and 18S sequences from all the Lake Malawˆ i the infectivity of Lake Malawi strains of S. haematobium isolates recovered from snails were identical, showing no to B. nyassanus, it is recommended that further compat- nuclear genetic variation. The phylogenetic analysis of the ibility studies are conducted to assess the possible 28S sequences showed that there is very little variation risk of the spread of schistosomiasis transmission to between the S. haematobium isolates incorporated into the other areas. tree, and the isolates from Lake Malawˆ i match the Preliminary mitochondrial genome data from different Zanzibar strain of S. haematobium (fig. 1). strains of S. haematobium have suggested that surprisingly In all mitochondrial DNA sequences, there was very little genetic variation occurs between different strains little variation between the Lake Malawˆ i isolates (table 3). and even between strains from very distant geographical All nucleotide differences were also non-synonymous, areas (Littlewood, Webster & Huyse, pers. comm.). In this suggesting that there is no genetic difference between study, we amplified, together with some conserved the different isolates. The phylogenetic analysis of the regions, the most variable regions of the mitochondrial COX1 sequences shows that there is very little variation genomes, aiming to capture any genetic variation that between the different S. haematobium isolates incorpor- might be occurring between the isolates. We postulated ated into the tree. The Malawˆ i isolates clearly group that the miracidia collected from children in Chembe with other S. haematobium strains, but there is not Village would infect both B. globosus and B. nyassanus, enough genetic variation between the isolates to give since we have collected infected individuals of both snail clear resolution (fig. 2). species on Nankumba Peninsula. Although we have In the first exposure experiment, 13 B. globosus were examined some 700 B. nyassanus (3.0–9.1 m depth) from infected from eggs that were captured from Chembe sites on Likoma Island and sites along the mainland Village children, 10 B. globosus were infected from eggs that shore in the northern part of the lake for cercarial were captured from Likoma Island, and no B. nyassanus shedding, we have not found a single individual that was were infected. When we repeated the exposure of B. infected. Based on these data, we concluded that all of the nyassanus to miracidia from eggs captured from Likoma urinary schistosomiasis found in inhabitants of Likoma Island children, one snail was infected. Island originated from cercariae shed from B. globosus. Therefore, it is significant that we were able to infect Discussion one B. nyassanus with eggs that originated from children from Likoma Island. The fact that we exposed 50 The lack of genetic variation between the S. haematobium B. nyassanus collected from Chembe Village, and only isolates from the two different intermediate snail hosts had one infected snail is consistent with the field observa- tions that showed that of 24,775 B. nyassanus collected, only 87 (0.4%) were infected (Stauffer et al., 2006). The fact Table 3. Mitochondrial DNA nucleotide differences of that the S. haematobium from Lake Malawˆ i can utilize S. haematobium isolates from different intermediate hosts and localities (number of single nucleotide polymorphisms) (accession Bulinus species from both the B. truncatus/tropicus and numbers EU567124–EU567144) compared to the Likoma isolate. the B. africanus group is somewhat unique. Furthermore, the fact that miracidia isolated from children at Likoma Sample area and host COX1 16S ND1 Pro-COX1 Island, where the only infected snails found were B. globosus, suggests that the Lake Malawˆ i S. haematobium Chembe, B. globosus always had the potential to infect both B. globosus and 10000B. nyassanus. 21233 30000Certainly, along the lake shore, where both B. globosus Chembe, B. nyassanus and B. nyassanus shed cercariae, the prevalence of 10010infection in school-aged children is 2–3 times higher 20001than where only B. globosus is a host (Stauffer et al., 2006). 30000Our current studies suggest that only in the waters Likoma, B. globosus of Nankumba Peninsula have population numbers of 10000B. nyassanus reached a critical level in shallow waters, 20000enabling it to become involved in the schistosome 30000 life cycle. Snail host susceptibility 381

Fig. 2. Neighbour joining (distance tree); COX1 (mitochondrial DNA).

Acknowledgements returning to Britain from the Tropics: a controlled study. Lancet i, 86–88. Partial funding was provided by the National Science Jørgensen, A., Jørgensen, L.V.G., Kristensen, T.K., Foundation (USA)/National Institutes of Health (USA) Madsen, H. & Stothard, J.R. (2007) Molecular joint programme in ecology of infectious diseases phylogenetic investigations of Bulinus (: (DEB0224958). ) in Lake Malawˆ i with comments on the topological incongruence between DNA loci. Zoologica Scripta 36, 577–585. References Madsen, H., Bloch, P., Kristensen, T.K. & Furu, P. (2001) Brown, D.S. (1994) Freshwater snails of Africa and Bulinus nyassanus is intermediate host for Schistosoma their medical importance. 2nd edn. London, Taylor & haematobium in Lake Malawˆ i. Annals of Tropical Francis. Medicine and Hygiene 95, 353–360. Cetron, M.S., Chitsulo, L., Sullivan, J.J., Pilcher, J., Pollner, J.H., Schwartz, A., Kobrine, A. & Parenti, D.M. Wilson, M., Noh, J., Tsang, V.C., Hightower, A.W. & (1994) Cerebral schistosomiasis caused by Schistosoma Addiss, D.G. (1996) Schistosomiasis in Lake Malawi. haematobium: case-report. Clinical Infectious Diseases 18, Lancet 348, 1274–1278. 354–357. Harries, A.D., Fryatt, R., Walker, J., Chiodini, P.L. & Rollinson, D., Stothard, J.R. & Southgate, V.R. (2001) Bryceson, A.D. (1986) Schistosomiasis in expatriates Interactions between intermediate snail hosts of the 382 J.R. Stauffer et al.

genus Bulinus and schistosomes of the Schistosoma Schistosomiasis in Lake Malawˆ i: relationship of fish haematobium group. Parasitology 123, S245–S260. and intermediate host density to prevalence of human Southgate, V.R., de-Clercq, D., Sene, M., Rollinson, D., infection. EcoHealth 3, 22–27. Ly, A. & Vercruysse, J. (2000) Observations on the Stothard, J.R., Bremond, P., Andriamaro, L., Sellin, B., compatibility between Bulinus spp. and Schistosoma Sellin, E. & Rollinson, D. (2001) Bulinus species on haematobium in the Senegal River basin. Annals of Madagascar: molecular evolution, genetic markers Tropical Medicine and Parasitology 94, 157–164. and compatibility with Schistosoma haematobium. Stauffer, J.R. Jr, Arnegard, M.E., Cetron, M., Sullivan, Parasitology 123, S261–S275. J.J., Chitsulo, L.A., Turner, G.F., Chiotha, S. & Whitworth, J.A.G. (1993) Schistosomiasis - may be contracted through swimming in Lake Malawˆ i. British McKaye, K.R. (1997) The use of fish predators to Medical Journal 307, 936. control vectors of parasitic disease: schistosomiasis in Lake Malawˆ i — a case history. BioScience 47, 41–49. (Accepted 9 July 2008) Stauffer, J.R. Jr, Madsen, H., McKaye, K., Konings, A., First Published Online 28 August 2008 Bloch, P., Fereri, C., Likongwe, J. & Makaula, P. (2006) q 2008 Cambridge University Press